RESUMO
RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence. X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues, and the consensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentrations equal to the enzyme concentration. This was confirmed by electron paramagnetic resonance of the enzyme reconstituted with manganese and crystal structures liganded with Mn2+ and a variety of sugar phosphates. Two transient species prior to the formation of products were identified using acid quench of single turnover reactions in combination with NMR for singly and fully 13C-labeled d-Ru5P. These data indicate that dehydration of C1 forms the first transient species, which undergoes rearrangement by a 1,2 migration, fusing C5 to C3 and generating a hydrated C4 that is poised for elimination as formate. Structures determined from time-dependent Mn2+ soaks of VcRibB-d-Ru5P crystals show accumulation in crystallo of the same intermediates. Collectively, these data reveal for the first time crucial transient chemical states in the mechanism of RibB.
Assuntos
Transferases Intramoleculares , Riboflavina , Butanonas , Formiatos , Transferases Intramoleculares/química , Fosfatos , Riboflavina/biossíntese , Riboflavina/química , Riboflavina Sintase/químicaRESUMO
Lumazine synthase (LS) is a family of enzyme involved in the penultimate step in the biosynthesis of riboflavin. Its enzymatic mechanism has been well defined, and many LS structures have been solved using X-ray crystallography or cryoelectron microscopy. LS is composed of homooligomers, which vary in size and subunit number, including pentamers, decamers, and icosahedral sixty-mers, depending on its species of origin. Research on LS has expanded beyond the initial focus on its enzymatic function to properties related to its oligomeric structure and exceptional conformational stability. These attributes of LS systems have now been repurposed for use in various biomedical fields. This review primarily focuses on the applications of LS as a flexible vaccine presentation system. Presentation of antigens on the surface of LS results in a high local concentration of antigens displayed in an ordered array. Such repetitive structures enable the cross-linking of B-cell receptors and result in strong immune responses through an avidity effect. Potential issues with the use of this system and corresponding solutions are also discussed with the objective of improved utilization of the LS system in vaccine development.
Assuntos
Sistemas de Liberação de Medicamentos/tendências , Complexos Multienzimáticos/administração & dosagem , Complexos Multienzimáticos/imunologia , Riboflavina Sintase/administração & dosagem , Riboflavina Sintase/imunologia , Animais , Sistemas de Liberação de Medicamentos/métodos , Humanos , Imunogenicidade da Vacina/imunologia , Complexos Multienzimáticos/química , Estrutura Secundária de Proteína , Riboflavina Sintase/químicaRESUMO
Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, with yields up to 40%. The fermentation products can subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes, FMN and FAD, by in vitro biotransformation with better than 90% yield. Using this approach, more than 100 single or multiple (13)C-, (15)N-, (17)O-, and (18)O-labeled isotopologues of these cofactors and ligands become easily accessible, enabling advanced ligand-based spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution.
Assuntos
Bacillus subtilis/química , Escherichia coli/química , Escherichia coli/enzimologia , Flavoproteínas/química , Marcação por Isótopo/métodos , Pteridinas/química , Pteridinas/síntese química , Purinas/química , Riboflavina Sintase/química , Riboflavina/química , Biotransformação , Ligantes , Riboflavina Sintase/metabolismoRESUMO
Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.
Assuntos
Brucella abortus/enzimologia , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Riboflavina/química , Riboflavina Sintase/genética , Homologia de Sequência de AminoácidosRESUMO
The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral ß subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.
Assuntos
Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Archaea/enzimologia , Bactérias/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fungos/enzimologia , Plantas/enzimologia , Pteridinas/metabolismo , Riboflavina/biossíntese , Schizosaccharomyces/química , Schizosaccharomyces/metabolismoRESUMO
Cofactors play key roles in metabolic pathways. Among them F(420) has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F(0)-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F(0)-synthase and their in vitro activities. Our study allows us to establish that F(0)-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F(0)-synthase generates two 5'-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes.
Assuntos
Actinomycetales/enzimologia , Mathanococcus/enzimologia , Nostoc/enzimologia , Riboflavina Sintase/metabolismo , Riboflavina/análogos & derivados , Tirosina/metabolismo , Actinomycetales/química , Actinomycetales/metabolismo , Mathanococcus/química , Mathanococcus/metabolismo , Nostoc/química , Nostoc/metabolismo , Estrutura Terciária de Proteína , Riboflavina/química , Riboflavina/metabolismo , Riboflavina Sintase/química , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismoRESUMO
Accurate structure-based sequence alignments of distantly related proteins are crucial in gaining insight about protein domains that belong to a superfamily. The PASS2 database provides alignments of proteins related at the superfamily level and are characterized by low sequence identity. We thus report an automated, updated version of the superfamily alignment database known as PASS2.4, consisting of 1961 superfamilies and 10,569 protein domains, which is in direct correspondence with SCOP (1.75) database. Database organization, improved methods for efficient structure-based sequence alignments and the analysis of extreme distantly related proteins within superfamilies formed the focus of this update. Alignment of family-specific functional residues can be realized using such alignments and is shown using one superfamily as an example. The database of alignments and other related features can be accessed at http://caps.ncbs.res.in/pass2/.
Assuntos
Bases de Dados de Proteínas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas/classificação , Riboflavina Sintase/químicaRESUMO
Riboflavin synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of riboflavin synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
Assuntos
Pteridinas/química , Riboflavina Sintase/química , Ânions/química , Sítios de Ligação , Biocatálise , Estrutura Molecular , Riboflavina/síntese química , Riboflavina/química , Riboflavina Sintase/metabolismo , Estereoisomerismo , Uridina/análogos & derivados , Uridina/síntese química , Uridina/químicaRESUMO
Riboflavin (vitamin B2) is the direct precursor of redox enzyme cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential for multiple cell physiology. The riboflavin biosynthetic pathway is regarded as a rich resource for therapeutic targets for broad spectrum antibiotics. Enzymatic pathways, regulatory factors of the riboflavin biosynthesis, and relevant drug discovery are summarized in this review. The novel riboswitch regulatory mechanism of riboflavin metabolism is also described. A compendium of chemical modulators of riboflavin biosynthesis and regulatory networks is listed and such demonstrates the promise of riboflavin biosynthesis and regulatory mechanisms as potential therapeutic targets for novel antibiotic drug discovery.
Assuntos
Anti-Infecciosos/farmacologia , Riboflavina Sintase/metabolismo , Riboflavina/biossíntese , Sequência de Aminoácidos , Anti-Infecciosos/química , Antituberculosos/química , Antituberculosos/farmacologia , Dados de Sequência Molecular , Riboflavina/metabolismo , Riboflavina Sintase/antagonistas & inibidores , Riboflavina Sintase/química , Alinhamento de SequênciaRESUMO
The pathway of riboflavin (vitamin B2) biosynthesis is significantly different in archaea, eubacteria, fungi and plants. Specifically, the first committed intermediate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, can either undergo hydrolytic cleavage of the position 2 amino group by a deaminase (in plants and most eubacteria) or reduction of the ribose side chain by a reductase (in fungi and archaea). We compare 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases from the yeast Candida glabrata, the archaeaon Methanocaldococcus jannaschii and the eubacterium Aquifex aeolicus. All three enzymes convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, as shown by 13C-NMR spectroscopy using [2,1',2',3',4',5'-13C6]2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate as substrate. The beta anomer was found to be the authentic substrate, and the alpha anomer could serve as substrate subsequent to spontaneous anomerisation. The M. jannaschii and C. glabrata enzymes were shown to be A-type reductases catalysing the transfer of deuterium from the 4(R) position of NADPH to the 1' (S) position of the substrate. These results are in agreement with the known three-dimensional structure of the M. jannaschii enzyme.
Assuntos
Archaea/enzimologia , Fungos/enzimologia , Riboflavina Sintase/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Riboflavina Sintase/química , Riboflavina Sintase/genética , Homologia de Sequência de Aminoácidos , Estereoisomerismo , UltracentrifugaçãoRESUMO
Icosahedral macromolecules have a wide spectrum of potential nanotechnological applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150 A icosahedral capsid consisting of 60 subunits and crystallizes in space group P6(3)22 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4 A resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77 A, alpha = beta = 90.0, gamma = 120 degrees , and diffracts to 1.6 A resolution.
Assuntos
Complexos Multienzimáticos/normas , Engenharia de Proteínas/normas , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/normas , Cristalização/métodos , Cristalização/normas , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida/métodos , Mutagênese Sítio-Dirigida/normas , Engenharia de Proteínas/métodos , Riboflavina Sintase/química , Riboflavina Sintase/genética , Riboflavina Sintase/normasRESUMO
The intensely fluorescent lumazine protein is believed to be involved in the bioluminescence of certain marine bacteria. The sequence of the catalytically inactive protein resembles that of the enzyme riboflavin synthase. Its non-covalently bound fluorophore, 6,7-dimethyl-8-ribityllumazine, is the substrate of this enzyme and also the committed precursor of vitamin B(2). An extensive crystallization screen was performed using numerous single-site mutants of the lumazine protein from Photobacterium leiognathi in complex with its fluorophore and with riboflavin, respectively. Only the L49N mutant in complex with riboflavin yielded suitable crystals, allowing X-ray structure determination to a resolution of 2.5 A. The monomeric protein folds into two closely similar domains that are structurally related by pseudo-C(2) symmetry, whereby the entire domain topology resembles that of riboflavin synthase. Riboflavin is bound to a shallow cavity in the N-terminal domain of lumazine protein, whereas the C-terminal domain lacks a ligand.
Assuntos
Proteínas de Bactérias/química , Proteínas Luminescentes/química , Photobacterium/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Evolução Molecular , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Óptica e Fotônica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Riboflavina/metabolismo , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Eletricidade EstáticaRESUMO
The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The final step in the biosynthesis of the vitamin involves the dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase. The mechanistically unusual reaction involves the transfer of a four-carbon fragment between two identical substrate molecules. The second product, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, is recycled in the biosynthetic pathway by 6,7-dimethyl-8-ribityllumazine synthase. This article will review structures and reaction mechanisms of riboflavin synthases and related proteins up to 2007 and 122 references are cited.
Assuntos
Riboflavina Sintase/metabolismo , Riboflavina/biossíntese , Sequência de Aminoácidos , Archaea/metabolismo , Bactérias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Riboflavina/química , Riboflavina Sintase/química , Homologia de Sequência de AminoácidosRESUMO
Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin. To obtain structural and mechanistic probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a sulfur analogue of the pyrimidine substrate of the lumazine synthase-catalyzed reaction and product of the riboflavin synthase-catalyzed reaction was designed. Facile syntheses of the S-nucleoside 5-amino-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione hydrochloride (15) and its nitro precursor 5-nitro-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione (14) are described. These compounds were tested against lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. Compounds 14 and 15 were found to be inhibitors of both riboflavin synthase and lumazine synthase. Compound 14 is an inhibitor of Bacillus subtilis lumazine synthase (Ki 26 microM), Schizosaccharomyces pombe lumazine synthase (Ki 2.0 microM), Mycobacterium tuberculosis lumazine synthase (Ki 11 microM), Escherichia coli riboflavin synthase (Ki 2.7 microM), and Mycobacterium tuberculosis riboflavin synthase (Ki 0.56 muM), while compound 15 is an inhibitor of B. subtilis lumazine synthase (Ki 2.6 microM), S. pombe lumazine synthase (Ki 0.16 microM), M. tuberculosis lumazine synthase (Ki 31 microM), E. coli riboflavin synthase (Ki 47 microM), and M. tuberculosis riboflavin synthase (Ki 2.5 microM).
Assuntos
Inibidores Enzimáticos/síntese química , Complexos Multienzimáticos/antagonistas & inibidores , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/farmacologia , Riboflavina Sintase/antagonistas & inibidores , Bacillus subtilis/enzimologia , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/enzimologia , Nucleosídeos de Pirimidina/metabolismo , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Especificidade por SubstratoRESUMO
3,4-Dihydroxy-2-butanone 4-phosphate synthase, 6,7-dimethyl-8-ribityllumazine synthase, and riboflavin synthase of the riboflavin biosynthetic pathway are potential targets for novel antiinfective drugs. This article describes a platform for high-throughput screening for inhibitors of these enzymes. The assays can be monitored photometrically and have been shown to be robust, as indicated by Z factors 0.87. A (13)C NMR assay for hit verification of 3,4-dihydroxy-2-butanone 4-phosphate synthase inhibitors is also reported.
Assuntos
Bacillus subtilis/enzimologia , Técnicas Analíticas Microfluídicas/métodos , Riboflavina Sintase/metabolismo , Riboflavina/antagonistas & inibidores , Riboflavina/biossíntese , Anti-Infecciosos/metabolismo , Bacillus subtilis/genética , Vias Biossintéticas , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Estrutura Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Peptídeo Sintases/antagonistas & inibidores , Pteridinas , Riboflavina/química , Riboflavina Sintase/químicaRESUMO
Riboflavin synthase from Escherichia coli is a homotrimer of 23.4 kDa subunits and catalyzes the formation of one molecule each of riboflavin and 5-amino-6-ribitylamino- 2,4(1H,3H)-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrate, 6,7- dimethyl-8-ribityllumazine. Each subunit comprises two closely similar folding domains. Recombinant expression of the N-terminal domain is known to provide a c(2)-symmetric homodimer. In this study, the binding properties of wild type as well as two mutated proteins of N-terminal domain of riboflavin synthase with various ligands were tested. The replacement of the amino acid residue A43, located in the second shell of riboflavin synthase active center, in the recombinant N-terminal domain dimer reduces the affinity for 6,7-dimethyl-8-ribityllumazine. The mutation of the amino acid residue C48 forming part of activity cavity of the enzyme causes significant (19)F NMR chemical shift modulation of trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine in complex with the protein, while substitution of A43 results in smaller chemical shift changes.
Assuntos
Escherichia coli/enzimologia , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Estereoisomerismo , Especificidade por Substrato , TitulometriaRESUMO
The penultimate step in the biosynthesis of riboflavin (vitamin B2) involves the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase). Pathogenic Brucella species adapted to an intracellular lifestyle have two genes involved in riboflavin synthesis, ribH1 and ribH2, which are located on different chromosomes. The ribH2 gene was shown previously to specify a lumazine synthase (type II lumazine synthase) with an unusual decameric structure and a very high Km for 3,4-dihydroxy-2-butanone 4-phosphate. Moreover, the protein was found to be an immunodominant Brucella antigen and was able to generate strong humoral as well as cellular immunity against Brucella abortus in mice. We have now cloned and expressed the ribH1 gene, which is located inside a small riboflavin operon, together with two other putative riboflavin biosynthesis genes and the nusB gene, specifying an antitermination factor. The RibH1 protein (type I lumazine synthase) is a homopentamer catalyzing the formation of 6,7-dimethyl-8-ribityllumazine at a rate of 18 nmol mg(-1) min(-1). Sequence comparison of lumazine synthases from archaea, bacteria, plants, and fungi suggests a family of proteins comprising archaeal lumazine and riboflavin synthases, type I lumazine synthases, and the eubacterial type II lumazine synthases.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Complexos Multienzimáticos/metabolismo , Riboflavina Sintase/metabolismo , Riboflavina/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Fases de Leitura Aberta , Filogenia , Riboflavina Sintase/química , Riboflavina Sintase/genética , Alinhamento de SequênciaRESUMO
Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers.
Assuntos
Archaea/enzimologia , Pteridinas/química , Ribitol/análogos & derivados , Riboflavina Sintase/química , Sequência de Aminoácidos , Archaea/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Cinética , Methanobacteriaceae/metabolismo , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Conformação Proteica , Estrutura Secundária de Proteína , Pteridinas/metabolismo , Ribitol/química , Ribitol/metabolismo , Riboflavina/química , Ribose/química , Homologia de Sequência de Aminoácidos , EstereoisomerismoRESUMO
The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The dismutation of 6,7-dimethyl-8-ribityllumazine catalysed by riboflavin synthase produces riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. A pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazines has been identified earlier as a catalytically competent reaction intermediate of the Escherichia coli enzyme. Acid quenching of reaction mixtures of riboflavin synthase of Methanococcus jannaschii, devoid of similarity to riboflavin synthases of eubacteria and eukaryotes, afforded a compound whose optical absorption and NMR spectra resemble that of the pentacyclic E. coli riboflavin synthase intermediate, whereas the CD spectra of the two compounds have similar envelopes but opposite signs. Each of the compounds could serve as a catalytically competent intermediate for the enzyme by which it was produced, but not vice versa. All available data indicate that the respective pentacyclic intermediates of the M. jannaschii and E. coli enzymes are diastereomers. Whereas the riboflavin synthase of M. jannaschii is devoid of similarity with those of eubacteria and eukaryotes, it has significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalysing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor.
Assuntos
Archaea/enzimologia , Bactérias/enzimologia , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Especificidade por Substrato , Tricosantina/químicaRESUMO
A synthetic gene specifying the catalytic domain of the Arabidopsis thaliana riboflavin synthase was expressed with high efficiency in a recombinant Escherichia coli strain. The recombinant pseudomature protein was shown to convert 6,7-dimethyl-8-ribityllumazine into riboflavin at a rate of 0.027 s-1 at 25 degrees C. The protein sediments at a rate of 3.9 S. Sedimentation equilibrium analysis afforded a molecular mass of 67.5 kDa, indicating a homotrimeric structure, analogous to the riboflavin synthases of Eubacteria and fungi. The protein binds its product riboflavin with relatively high affinity (Kd =1.1 microM). Product inhibition results in a characteristic sigmoidal velocity versus substrate concentration relationship. Characterization of the enzyme/product complex by circular dichroism and UV absorbance spectroscopy revealed a shift of the absorption maxima of riboflavin from 370 and 445 to 399 and 465 nm, respectively. Complete or partial sequences for riboflavin synthase orthologs were analyzed from 11 plant species. In each case for which the complete plant gene sequence was available, the catalytic domain was preceded by a sequence of 1-72 amino acid residues believed to function as plastid targeting signals. Comparison of all available riboflavin synthase sequences indicates that hypothetical gene duplication conducive to the two-domain architecture occurred very early in evolution.